T-lineage acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic cancer in which novel more effective antileukemic drugs are needed for the treatment of patients with chemotherapy resistant disease. We recently found that aberrant activation of the NOTCH signaling pathway plays a critical role in the pathogenesis of more than 50% of human T-cell lymphoblastic leukemias (T-ALL), in which activating mutations in NOTCH1 result in constitutive NOTCH signaling. Thus, inhibition of NOTCH 1 signaling using y-secretase inhibitors (GSIs) to block a critical activation-associated proteolytic cleavage of the NOTCH1 receptor is emerging as a promising novel strategy for the treatment of T-ALL. Our central hypothesis is that the aberrant activation of NOTCH1 regulates critical downstream target genes which contribute to cell transformation in T-ALL. The goals of this research are to identify specific pathogenic target genes, transcriptional programs and molecular mechanisms that lie downstream of oncogenic NOTCH1, and assess their contribution to the transformation of T-cell progenitors. Ultimately, we will exploit these results to develop effective, highly specific therapies based on molecularly targeted antileukemic agents. To achieve these objectives, we propose the following specific aims: Aim 1. To define the structure of the transcriptional programs activated by NOTCH1 in T-ALL. In this aim, we will use a combination of gene expression arrays and ChlP-on-chip analysis to define the structure of the transcriptional network activated by aberrant NOTCH1 signaling. Aim 2. To clarify the specific leukemogenic mechanisms by which aberrant activation of NOTCH1 signaling in T-cell progenitors results in dysregulated proliferation and survival. This specific aim is based on the hypothesis that the transcriptional program activated by the constitutive NOTCH1 signaling contributes to the pathogenesis of T-ALL through the disordered regulation of cell proliferation and apoptosis in the context of T-cell development. Thus, we will define the specific roles and the mechanisms through which each of these pathways contributes to the pathogenesis of NOTCH- induced thymic malignancies. To achieve this goal we will block NOTCH1 signaling at strategic points in T-ALL cells and determine the mechanisms that mediate the effects and of inactivation of this oncogene in: (i) cell cycle progression, (ii) induction of apoptosis and (iii) tumorigenesis.